The goal of this research is to elucidate the structure of the prion protein in its aggregated isoforms, and to elucidate the relationship between isoform structure and different types of prion diseases. Prion protein is the causative agent of many fatal neurodegenerative diseases of mammals. These diseases are characterized by the conversion of normal, monomeric prion protein (PrPC) to a misfolded conformational state that aggregates and accumulates as fibrillar plaques in the brain. Transmission of the disease can occur when one mammal ingests the infected tissue of another. When exposed to an infectious aggregate, normal PrPC takes on the misfolded conformation and the fibrils are thus propagated. Any treatment of a prion disease must prevent or abrogate misfolding/aggregation while not affecting normal PrP function. Hence, it is important to understand the structural differences between the PrP isofoms. High resolution structures of PrPC from many mammals have been determined by both NMR and X-ray crystallography. In contrast, the insoluble, fibrillar nature of the infectious form has hampered the elucidation of its structural details. However, numerous biophysical and spectroscopic studies suggest that the misfolded form contains a much higher proportion of -sheet than normal PrP, which is mostly a-helical/random coil. This work will elucidate the nature of the PrP subunit interactions in several forms of recombinant PrP by using two biophysical techniques: site directed spin labeling (SDSL) combined with electron paramagnetic spectroscopy (EPR) and site directed crosslinking combined with mass spectrometry (MS). The EPR experiments provide information on protein backbone dynamics, secondary, tertiary, and quaternary structure, distances between labeled residues, and the mobility of the label itself. The crosslinking experiments will confirm the structural information gleaned from EPR. PUBLIC HEALTH RELEVANCE The elucidation of the details of the structure of prion fibrils will be of great value in developing treatments for prion diseases, which are fatal neurodegenerative diseases of mammals. These include Creutzfeldt-Jakob disease (CJD) and variant CJD, Gerstmann- Straussler-Scheinker disease, Fatal Familial Insomnia, and kuru (humans), scrapie (sheep), chronic wasting disease (elk, deer), and mad cow disease (cattle). Treatment of any prion disease must prevent or abrogate fibril formation while not affecting normal prion function; hence, it is important to understand the structural differences among the different prion isofoms.